How probiotics, prebiotics, synbiotics, and postbiotics prevent dental caries: an oral microbiota perspective

Dental caries, a highly prevalent oral disease, impacts a significant portion of the global population. Conventional approaches that indiscriminately eradicate microbes disrupt the natural equilibrium of the oral microbiota. In contrast, biointervention strategies aim to restore this balance by introducing beneficial microorganisms or inhibiting cariogenic ones. Over the past three decades, microbial preparations have garnered considerable attention in dental research for the prevention and treatment of dental caries. However, unlike related pathologies in the gastrointestinal, vaginal, and respiratory tracts, dental caries occurs on hard tissues such as tooth enamel and is closely associated with localized acid overproduction facilitated by cariogenic biofilms. Therefore, it is insufficient to rely solely on previous mechanisms to delineate the role of microbial preparations in the oral cavity. A more comprehensive perspective should involve considering the concepts of cariogenic biofilms. This review elucidates the latest research progress, mechanisms of action, challenges, and future research directions regarding probiotics, prebiotics, synbiotics, and postbiotics for the prevention and treatment of dental caries, taking into account the unique pathogenic mechanisms of dental caries. With an enhanced understanding of oral microbiota, personalized microbial therapy will emerge as a critical future research trend.

with the use of probiotics, prebiotics, synbiotics, and postbiotics in the prevention of dental caries.Lastly, this article proposes future directions for the development of this field to provide more scientific, standardized, and effective guidelines for the prevention of dental caries from an academic perspective.

Dental caries Background
The oral cavity is a complex ecosystem characterized by various warm, moist microenvironments that provide ideal conditions for microbial growth 7 .A recent study analyzing the oral microbiome identified a total of 1591 microbial species, including bacteria, fungi, archaea, viruses, and protozoa 8 , second only in complexity to the colon 9 .The core oral microbiota in healthy individuals remains relatively stable over seven years 10 , while an imbalanced oral microbiota can lead to dental caries and other oral diseases 11 .Moreover, the diversity of the oral microbial community in severe dental caries is considerably lower than that in healthy individuals 12 .Dental caries arises from an imbalance in the oral microbiota resulting from a complex interplay between the host, diet, and microorganisms 13 .
Of these factors, fermentable carbohydrates, which are commonly found in sweetened foodstuffs, have been identified as particularly important dietary contributors to dental caries 14 .Consumption of sweetened foodstuffs can rapidly increase the concentration of carbohydrates in the oral cavity, leading to a sharp decline in the pH values of biofilm to 4 or even lower 15 .Research has revealed a precise correlation between the areas of acute demineralization on the enamel surface and the highly acidic pH zones created by biofilms 16 .This is attributed to the frequent local pH decreases can disrupt the balance between tooth mineralization and demineralization in the closed microenvironment of the biofilm 17 .Consequently, this leads to mineral loss in teeth, resulting in white spots, cavitation, pulp infections, and even tooth loss 17 .
Differences in oral microflora have been observed between individuals with healthy teeth and those with dental caries.For example, the findings of a study on the oral microbiome of children indicate that the genera Rothia, Neisseria, and Haemophilus, which are among the first colonizers of the oral cavity following birth 18 , are associated with dental health 19 .In contrast, Prevotella spp., S. mutans, and Human herpesvirus 4 (EB virus) are more commonly found in children with dental caries 19 .Actinomycetota (35.8%) and Bacillota (31.2%) were the most common phyla in deep dentin carious lesions, and Lactobacillus was the most abundant genus in only 25% of the carious lesions 20 .There is increasing recognition that dental caries is caused by the imbalanced microbiota in the biofilm, also known as dental plaque, rather than by a single pathogen 21 .

Microorganisms associated with dental caries
The cariogenic bacteria exhibit varying degrees of contribution to the development of dental caries.For decades, S. mutans and Streptococcus sobrinus have been widely recognized as the major cariogenic agents 22 .It is noteworthy that S. sobrinus exhibited superior acidogenicity and aciduricity compared to S. mutans, but showed lesser adaptability to the biofilm environment 23 .The cariogenic bacteria within the oral microbiota do not exist as isolated entities but rather interact and influence each other.To a certain extent, S. mutans creates a lactic acid-rich environment in carious lesions that facilitates the proliferation of Veillonella species 12 , which have been shown to promote the growth of S. mutans in biofilm studies 24 .
Additionally, Candida species, as a typical fungal representative of cariogenic microorganisms, have emerged as potent secondary cariogenic agents, isolated from 40% to 60% of adult and pediatric caries 25 .Candida is a powerful opportunistic caries yeast that relies on the production of shortchain carboxylic acids and proteinases, as well as its ability to adhere to abiotic surfaces and form biofilm 25 .The most common communication between fungi and bacteria in the oral cavity is the mutual interaction between Candida albicans and S. mutans.The presence of C. albicans promotes the growth of S. mutans, eliciting notable changes in gene expression and enhancing carbohydrate metabolism 26 .Notably, compared to the mono-species biofilm comprising solely S. mutans, there are 393 differentially expressed genes in S. mutans within the dual-species biofilm 26 .The glucosyltransferases (Gtf) secreted by S. mutans can bind to C. albicans and facilitate the conversion of sucrose into exopolysaccharide (EPS), thereby providing binding sites for S. mutans 27 .
One study reported that the core microbiota of early childhood caries (ECC) may include Veillonella parvula, Fusobacterium nucleatum, Prevotella denticola, and Leptotrichia wadei 28 .On the one hand, this ECC core microbiota promotes the growth and acidogenicity of S. mutans, and promotes biofilm formation, albeit with limited acidogenic capacity 28 .On the other hand, it also promotes enamel demineralization in vitro and increases the cariogenic potential of enamel in vivo 28 .Additionally, according to some metagenomic results, the following species are closely associated with dental caries: Streptococcus gordonii, Leptotrichia buccalis, V. parvula, Actinomyces gerencseriae, Propionibacterium acidifaciens, Hallella multisaccharivorax, and Parascardovia denticolens 29,30 .
Of the microorganisms associated with dental caries, S. mutans is one of the most extensively studied species in this field.Given that S. mutans was initially thought to be a major cause of dental caries 31 , it is not surprising that most prevention strategies target this bacterium specifically 29 .

Streptococcus mutans
When compared with other original colonizing bacteria, S. mutans exhibits more advantageous traits by developing a compact biofilm and its distinctive virulence factors 32 .Furthermore, under the regulated control of the quorum-sensing system, S. mutans ultimately becomes one of the major cariogenic bacteria.The biofilm in dental caries of primary teeth is a threedimensional (3D) spherical structure, with S. mutans as the core and other bacteria forming the outer layer 16 .This localized area creates an acidic pH environment, leading to severe enamel demineralization 16 .As dental caries progresses, the diversity of the oral microbiota becomes limited 33 .This microbial imbalance eventually leads to the occurrence and development of dental caries.The close association between S. mutans and dental caries has been confirmed.Although S. mutans is a natural resident of the human oral cavity 34 , an increase in the levels of S. mutans should be of concern as it may indicate a clinical precursor to dental caries 35 .Rats infected with humanderived S. mutans develop dental caries 36 , and S. mutans may be associated with severe-ECC recurrence 37 .These suggest the crucial role of S. mutans in the occurrence and development of dental caries.Therefore, further elucidating the pathogenic mechanisms of S. mutans (Fig. 1) is essential for the development of effective strategies for caries prevention and treatment.In the following sections, we will discuss the virulence factors and quorumsensing system aspect of S. mutans.
Virulence factors.The virulence factor of S. mutans can be categorized into four major groups, comprising EPS synthesis, adhesion, acid production, and acid resistance.
The synthesis of EPS.The ability of S. mutans to exert its pathogenicity is largely attributed to the production of EPS.EPS, a major component of biofilms 38 , consists of extracellular proteins, extracellular DNA, and lipoteichoic acid 39 .The primary component of EPS is glucan, which is synthesized by Gtf 40 , providing binding sites for microorganisms 38 .EPS contributes to the formation of highly organized chemical and physical barriers within the biofilm matrix, facilitating microbial adherence to non-living surfaces 41 , resisting fluid shear stresses 42 , evading host immune responses 43,44 , tolerating antimicrobial agents 45 , and ultimately establishing and maintaining acidic microenvironment in the oral cavity that favors the development of dental caries-associated biofilm communities 42 .Mature biofilms are difficult to remove mechanically due to the enhanced viscoelasticity conferred by EPS 42 .EPS may achieve immune evasion by mediating complement evasion 43 and limiting the entry of effector molecules from the innate and adaptive immune systems into the biofilm matrix 44 .Chlorhexidine, a commonly used antimicrobial agent in oral care, has limited penetration into deep biofilm layers due to its positive charge, whereas "the fuel" (sucrose), lacking charge, can easily diffuse, facilitated by the negative charge of EPS 45 .The negatively charged surface of S. mutans cells enveloped by EPS accumulates protons, and the sieving effect of the glucan structure also plays a role 46 .On one hand, EPS captures and accumulates protons produced externally or by acidogenic microorganisms, aiding in the retention and accumulation of acid within the biofilm 46 .On the other hand, once protons are recruited to the cell surface, they trigger an acid adaptation response, allowing the microorganisms to preemptively counteract acid damage 46 .Deactivation of one or more gtf Fig. 1 | Cariogenic biofilm formation.EPS exopolysaccharides, Gbps Glucanbinding proteins, gbp encode Gbps, be related to adhesion, Gtfs Glucosyltransferases, gtf encode Gtfs, be related to the synthesis of EPS, eno encode Bacteria enolase, be related to glucose uptake, LDH lactate dehydrogenase, ldh encode LDH, be related to acid production, PTS phosphotransferase, the glucose uptake system, PEP phosphoenolpyruvate, atpD encode F 1 F 0 -ATPase, be related to acid resistance, ATP adenosine triphosphate, ADP adenosine diphosphate, AgDs agmatine deiminase system, aguD encode AgDs, be related to acid resistance.Healthy teeth develop dental caries due to the complex interactions between the host, diet, and microorganisms.The microecology of healthy teeth is based on the balance between acidogenic and alkalinogenic microbial activities, as well as the balance between demineralization and remineralization processes.When acidogenic microorganisms become predominant, frequent and high concentrations of acid locally lead to net demineralization of dental enamel, resulting in the formation of cavities.At the micro level, the initial step involves the adhesion of some primitive colonizing microorganisms to the dental enamel.The second step involves the production of EPS by the microorganisms, forming a biofilm.In the third step, acidogenic and acid-tolerant microbial communities, mainly dominated by S. mutans, establish a highly acidic microenvironment, leading to demineralization of the dental enamel.In the fourth step, the highly acidic microenvironment confers a growth advantage to S. mutans-dominated microbial populations.Taking the virulence factors of S. mutans as an example, the synthesis of EPS is primarily mediated by Gtfs.The adhesion process is mainly facilitated by Gbps.Acid production involves the participation of enolase and LDH.Acid tolerance processes primarily rely on the involvement of F 1 F 0 -ATPase and AguD.
genes significantly reduces the virulence of S. mutans, in rodent caries models 47 .In summary, EPS plays a crucial role in enabling S. mutans to exert its cariogenic potential.Therefore, inhibiting EPS synthesis may represent a feasible preventive strategy against dental caries 48 .
Adhesion.S. mutans employs both sucrose-independent and sucrosedependent pathways to adhere to teeth 47 .The initial adhesion process is primarily mediated by the sucrose-independent pathway, which is subsequently reinforced by the stimulation of glucan synthesis via the sucrosedependent pathway, ultimately culminating in the formation of biofilms 47 .Glucan-binding proteins facilitate the binding of glucans synthesized from sucrose through glucose transferases.Of these proteins, GbpA exhibits a strong correlation with cariogenicity 49 .On the one hand, it contributes to the formation of strong biofilm structure and is an important protein determining the structure of biofilm.On the other hand, it plays an essential role in linking glucan molecules and is involved in the bacterial adhesion process to teeth.Acid production.After glucose metabolism, dietary carbohydrates produce energy and organic acids as metabolic by-products 50 .The acid-producing activity of S. mutans is not only a critical factor contributing to its pathogenicity but also a crucial characteristic leading to dental caries.Bacteria enolase, an enzyme encoded by the gene eno, is a primary component of the phosphotransferase system, which is responsible for glucose uptake 51 .Through the rapid catalytic activity of lactate dehydrogenase (LDH), a protein encoded by the ldh gene, S. mutans UA159 ferments glucose into organic acids 52 .
Acid resistance.S. mutans employs some acid-resistant mechanisms to cope with the stress of increasing acid production.F 1 F 0 -ATPase, a proton pump encoded by atpD 47 , not only pumps out intracellular protons to maintain intracellular pH but also produces ATP to promote bacterial growth and survival 53 .Inhibition of atpD expression in S. mutans UA159 resulted in a significant decrease in acid adaptation and an increase in cytoplasmic acidity 51 .Additionally, S. mutans produces alkali to neutralize acids, as well as export them out of the cells.The agmatine deiminase system plays a crucial role in producing alkalis to overcome acid stress 54 .Amongst its components, the agmatine-putrescine antiporter (AguD), encoded by the aguD gene, is of particular importance as it facilitates the intracellular transport of free agmatine 54 .The accumulation of protons on the surface of bacterial cells enveloped by EPS plays a significant role in the acid resistance of S. mutans, as mentioned in the "synthesis of EPS" section 46 .
Quorum-sensing (QS) system.The QS system regulates virulence and biofilm formation by releasing, sensing, and interacting with diffusion molecules 55 based on cell density in the surrounding environment 38 .S. mutans utilize this system to communicate with each other as a group rather than as separate individuals.The main mechanism for signal feedback is via the two-component signal transduction systems (TCSTS), which enable bacteria to regulate their gene expression 56 .S. mutans contains several types of TCSTS, among which VicRKX and ComCDE are critical in the regulation of biofilm formation, acid resistance, and acid production in response to environmental signals 57,58 .If these regulatory systems fail to function properly, it may lead to a decrease in the cariogenicity of S. mutans.

Dental caries prevention measures-biological interventions
Although plaque is a natural occurrence in teeth from an evolutionary, biological, and nutritional perspective, an imbalance in the microbiome of the oral pathological biofilm can lead to the development of dental caries 59 .Acid-producing cariogenic bacteria, especially S. mutans, damage the hard tooth structures in the presence of fermentable carbohydrates 38 .
In recent years, the field of biological intervention has developed some novel strategies.One approach involves using predators, such as Bdellovibrio, Bacteriovorax, and Peredibacter, to eliminate anaerobic Gram-negative bacteria that are periodontal pathogens 60,61 .Given that beneficial bacteria are mostly Gram-positive 62 .Additionally, biological interventions also include the use of specific inhibitors of S. mutans proteins, vaccination, and passive immunization strategies with neutralizing bacteria 25,29 .Although some innovative biological intervention strategies such as those mentioned above have emerged, the use of microbial preparations, such as probiotics, prebiotics, synbiotics, and postbiotics, is a more established and popular approach for preventing dental caries.

Probiotics Background
Probiotics were discovered by scholars as early as 1908 63 , and since then the field of studying the health effects of probiotics on the host has gradually developed.In 2013, The International Scientific Association of Probiotics and Prebiotics (ISAPP) defined probiotics as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" 64 .Today, probiotics are commonly used by humans to maintain their overall well-being.Although their effectiveness in promoting gastrointestinal health is well-known, research has also shown that probiotics can be effective in preventing and treating various oral diseases, such as dental caries, oral mucositis, and halitosis 65 .
In dentistry, probiotics were first introduced by Meurman and colleagues 6 , who found that Lacticaseibacillus rhamnosus GG ATCC 53103 could colonize the human mouth.With further research, probiotics have been found to have a remarkable ability to prevent dental caries.For instance, one study explored the effect of subjects' own Lactobacillus on S. mutans 66 .The study has shown that Lactobacillus isolated from the oral cavity of subjects can effectively inhibit the growth of S. mutans.The most effective species of S. mutans were found to be Lacticaseibacillus paracasei and Lactiplantibacillus plantarum, which are also the most common isolates.Finally, the use of probiotics in the treatment of oral diseases has been found to restore oral microbial balance and reduce the levels of S. mutans in dental plaque and saliva 67 .
With different probiotic strains exhibiting unique characteristics, understanding the specifics of each strain is crucial to when prevention and treatment of dental caries.For instance, L. rhamnosus GG is a homofermentative Lactobacillus that is not considered to be cariogenic because it cannot ferment sucrose or lactose 68 .Limosilactobacillus reuteri is an obligate heterofermentative species 68 that can produce broad-spectrum antimicrobials with good acid-base stability, such as reuterin 69 and reutericyclin 70 .In addition to Lactobacillus spp., Bifidobacteria spp.may also be a potential probiotic for preventing and treating dental caries.Yogurt containing Bifidobacterium DN-173010 has been reported to significantly reduce the level of S. mutans 71 .

Mechanisms to prevent dental caries
The mechanism through which probiotics can prevent dental caries is similar to that found in the gastrointestinal tract.The principal inhibitory mechanisms include the synthesis of active metabolites, inhibition of cariogenic microbial biofilm, competitive adhesion and colonization, coaggregation with pathogens, and regulation of the immune system (Fig. 2).

Production of active metabolites
Bacteriocin.Bacteriocin is a cationic antibacterial peptide synthesized by the ribosome 72 and was first discovered 73 .Bacteriocins can be divided into four different classes, with Classes I and II being the primary focus of most probiotics research 74 .Nisin, a bacteriocin of Type A in Class I, is widely recognized as a small positively charged protein (2-5 kDa) that induces target cells to form membrane pores 72 .Class II bacteriocins kill bacteria by increasing membrane permeability and leaking target bacterial contents 75 .
In addition to the above mechanisms, bacteriocins can also inhibit the synthesis of biofilm and cell wall, exert the activities of DNase and RNase, and regulate microbiota 72 .
Protein-protein interaction between the GtfB and LuxS proteins of S. mutans and bacteriocin of SD1 in L. paracasei was found to reduce the https://doi.org/10.1038/s41522-024-00488-7 Review article npj Biofilms and Microbiomes | (2024) 10:14 formation of biofilm and the density of microorganisms, as revealed in a simulation study 76 .Following comprehensive bioinformatics analysis and characterization, the bacteriocin in this study was found to be safe for humans.The bacteriocin Mersacidin exerts its bactericidal activity by forming a complex with lipid II, which inhibits cell wall synthesis 77 .The DNase and RNase activities of colicins from E2 to E9 enable them to nonspecifically degrade bacterial DNA and RNA 78 .Among these, colicin E2 exhibits potent and long-lasting bactericidal activity, and interestingly, it can specifically target bacteria in complex biological membranes 79 .Bacteriocins are capable of promoting the colonization of producer bacteria in specific niches over a decade, regulating the composition of the microbiota and affecting the host immune system 80 .Both nisin and nisin-producing probiotics can reduce the level of pathogens in biofilm and restore the diversity of strains to a healthy level 81 .
As bacteriocins are polypeptides and proteins, temperature control is crucial to ensure their activity during production and use.Streptococcus oralis subsp.dentisani 7746 (AB-Dentisanium ® ), for instance, is optimally concentrated at 30 and 45 °C, with a small reduction in bacteriocin activity at 60 °C82 .This critical consideration underscores the importance of implementing appropriate temperature regulation strategies in the development and use of bacteriocins for various applications.
Enzyme.In addition to bacteriocins, probiotics synthesize a diverse range of enzymes that confer beneficial effects by decomposing biofilms and affecting bacteriocin activity.For instance, Lactobacillus acidophilus can secrete lipase to degrade biofilm 83 .Similarly, Streptococcus salivarius JH expresses a dextranase enzyme that can hydrolyze the EPS of S. mutans and increase the anti-S.mutans inhibitory activity of zoocin A, a muralytic bacteriocin 84 .Another example is Streptococcus sp.A12, which produces challisin-like proteases that inhibit the production of bacteriocins by S. mutans 85 .Additionally, S. salivarius M18 produces urease and dextranase to neutralize salivary acidity and reduce plaque formation, respectively 86 .
Biosurfactants (BS).Biosurfactants are amphiphilic substances produced by microbial metabolism that contain both hydrophobic and hydrophilic groups, mainly composed of proteins, sugars, and lipids 87 .The structure of BS can be identified using various techniques such as thin layer chromatography, Fourier Transform Infrared Spectrometer, and Nuclear Magnetic Resonance 88 .Fifty percent of the 40 biosurfactant reports reviewed did not analyze their structure, likely due to the complexity of the structures 87 .Surfactin and/or protein-like biosurfactants (32.5%) are most commonly produced by Lactobacillus, with studies on glycoproteins (7.5%) 89 , glycolipid (5%) 90 , and glycolipopeptide (5%) 91 production being rare.Lactobacillus typically produces surfactin-type biosurfactants, which are protein-rich and can significantly inhibit pathogen adhesion, making them increasingly interesting due to their unique anti-adhesion and antibiofilm properties 87 .For example, BS produced by L. reuteri DSM 17938, L. acidophilus DDS-1, L. rhamnosus ATCC 53103, and L. paracasei B21060 inhibited S. mutans and Streptococcus oralis adhesion and biofilm formation in a dose-dependent manner on titanium surfaces 92 .In more detail, L. acidophilus DSM 20079 produces protein-type BS, which can shorten the chain length of S. mutans, interfere with its biofilm formation on glass slides, and down-regulate the gene expression of gtfB and gtfC 93 .BS produced by L. rhamnosus can destroy the physical structure or protein conformation of biofilm, leading to cell lysis 94 .In addition to the appealing antimicrobial activity mentioned above, BS exhibits characteristics of low cytotoxicity and high stability.BS derived from Lactobacillus spp.demonstrate comparable low cytotoxicity to rhamnolipids, which are generally regarded as non-toxic products 87 .BS may exhibit better stability compared to other antibacterial substances produced by probiotics.Gudinahe et al. 95 isolated stable BS from L. paracasei.This BS was pH stable within a range of 6-10 and maintained surface activity after incubation at 60 °C for 120 h.Organic acids.Organic acids, such as lactic acid and butyric acid 96 , produced by Lactobacillus in the human gastrointestinal tract and other body parts, have widely been recognized as beneficial substances.These organic acids may have a bacteriostatic effect on oral pathogenic microorganisms to a certain degree.For instance, L. paracasei Lpc-37 produces the acid that can restrain the growth and biofilm formation of S. mutans 97 .However, considering the strong association between dental demineralization and frequent exposure to high concentrations of acid 16 , it raises questions about how the acids produced by probiotics counteract the acids produced by cariogenic bacteria, including S. mutans.
These questions may need to be approached from the perspective of the overall caries environment.Cariogenic microorganisms create a highly organized acidic barrier 42 .Prolonged exposure to localized high concentrations of acid leads to localized demineralization rather than average demineralization of the teeth.If the organic acids produced by probiotics can inhibit cariogenic bacteria, including S. mutans, and/or their biofilms, they may disrupt this acidic barrier and prevent acid accumulation.Given the presence of its own acid-base microbial balance in the oral cavity 54 , acids that do not accumulate but instead contribute to the acid-base equilibrium in the oral environment appear to pose a lesser threat.
Hydrogen peroxide.Certain probiotic species, including Bifidobacterium bifidum, Lactobacillus johnsonii, Lactobacillus crispatus, and Lactobacillus jensenii, produce hydrogen peroxide to exert antibacterial effects 98 .Hydrogen peroxide acts on the pathogenic bacteria's epithelium, leading to their death 98 .Moreover, hydrogen peroxide has the potential to regulate species composition within the oral cavity 99 .Notably, cariogenic species such as S. mutans are highly susceptible to hydrogen peroxide toxicity 100 .However, the antibacterial activity of L. paracasei cell-free supernatant (CFS) was significantly reduced after catalase treatment, indicating the involvement of hydrogen peroxide in its bacteriostatic effects 101 .It should be noted that hydrogen peroxide may not retain its bacteriostatic function after processes such as freeze-drying, owing to the ease of its decomposition 102 .
Inhibition of cariogenic microbial biofilm.Dental caries is commonly mediated by biofilm.A crucial property of probiotics is the ability to inhibit or eliminate the growth of biofilms and pathogenic microorganisms in the oral cavity.Some strains have often been reported for their anti-biofilm activity, including S. oralis 89a, Limosilactobacillus fermentum TCUESC01, L. acidophilus 4A, and Bifidobacterium longum subsp.longum 103 .For instance, Lacticaseibacillus casei ATCC 393, L. reuteri ATCC 23272, L. plantarum ATCC 14917, and Ligilactobacillus salivarius ATCC 11741 may suppress the biofilms of S. mutans by downregulating genes such as gtfB, gtfC, and gtfD in S. mutans 104 .Interestingly, probiotics are capable of inhibiting fungi from transitioning into pathogenic forms.It has been demonstrated that L. rhamnosus LR32, L. casei L324m, and L. acidophilus NCFM exhibit the ability to impede the initial stages of hyphal formation, which is a crucial step in the pathogenesis of C. albicans 105 .A probiotic combination consisting of Lactobacillus helveticus CBS N116411, L. plantarum SD5870, and S. salivarius DSM 14685 significantly down-regulated the expression of genes involved in yeast-hypha transition in C. albicans, including EFG1 (hyphae-specific gene activator), SAP5 (secreted protease), ALS3 (adhesin/invasin) and HWP1 (hyphal wall protein) 106 .In vitro, biofilm models are continuously improving to replicate more closely the conditions found within the human body.Based on the specific research objectives, researchers can opt for models of interest, such as an experimental abutment mimicking the macro-and microstructure of a dental implant 107 .
The combination of L. rhamnosus and collagen peptides was found to significantly increase the pH of the medium in the early stages of biofilm formation 108 .The qPCR results showed that this combination downregulated several crucial genes linked to acid production and acid tolerance, including eno, ldh, and atpD.Moreover, probiotics may also exert antibacterial effects by interfering with QS.A study revealed that comD, vicR, and vicK genes were down-regulated in planktonic and biofilm forms of S. mutans when exposed to CFS with Lactobacillus 104 .This effect may explain the reduced adherence and biofilm formation of S. mutans observed in scanning electron microscopy experiments.
Competitive adhesion and colonization.One of the key characteristics of probiotics contributing to their health effects is their capacity for outcompeting oral pathogens concerning adhesion and colonization 109 .For example, L. reuteri LR6 displayed the most substantial adhesion capabilities among eight tested probiotic strains, which corresponded to a higher ability for inhibiting the adherence of pathogens to Caco-2 cells 110 .Enhanced colonization efficacy by S. salivarius M18 resulted in stronger anti-caries activity as evidenced by a reduction in plaque scores and S. mutans levels 111 .Levilactobacillus brevis KCCM 202399 inhibited the adherence of S. mutans KCTC 5458 by reducing the self-aggregation, cell surface hydrophobicity, and EPS production of S. mutans 112 .
Interestingly, probiotics can reduce pathogen adhesion even without direct contact.Saliva treated with probiotics was shown to reduce the adhesion of S. mutans to hydroxyapatite surfaces (a model for enamel) 113 .Further studies showed that the above salivary membrane lacked two proteins: salivary lectin gp340, the primary receptor for S. mutans in the salivary membrane, and salivary peroxidase, an innate defense factor found in human saliva 114 .
Coaggregation with pathogens.Coaggregation is among the advantageous properties of probiotics as it allows them to form a barrier that impedes pathogen colonization 115 .In a study, six out of 624 lactic acid bacteria were found to exhibit specific coaggregation with S. mutans in vitro 116 .These species were identified as L. paracasei and L. rhamnosus.It was discovered that this coaggregation mechanism is highly resilient to both hyperthermia and protease, and does not rely on lectins, nor is it impacted by saliva.
Regulation of the immune system.In addition to their direct effects on pathogenic microorganisms or biofilm, probiotics are known to activate and modulate the host's immune system 117 .Clinical studies have shown that daily or tri-weekly consumption of L. paracasei SD1 in patients with severe ECC significantly enhances salivary levels of human neutrophil peptides 1-3 with a broad bactericidal activity and reduces S. mutans levels, potentially slowing the progression of caries 118 .Furthermore, the consumption of milk containing L. paracasei SD1 for six months increased salivary immunoglobulin A levels, and this increase is positively correlated with a load of L. paracasei 119 .

The application vehicle
The colonization of probiotics in the oral cavity may be influenced by the choice of the delivery vehicle 68 .A range of vehicles is available for delivering probiotics, including dairy products, ice cream, cereal, pacifiers, chewing gum, curd, juice, and mouth wash (Table 1).Table 1 also mentions the test species, dose, and efficacies.
Among these vehicles, dairy products are considered ideal carriers due to their inherent beneficial characteristics 121 .Among the dairy products, liquid substrates, such as milk and yogurt, were found to be more effective in reducing S. mutans levels 122 .For individuals who are allergic to dairy products, alternative carriers may be selected, as illustrated in Table 1.The buffering capacity of milk helps to reduce acid production, while its colloidal nature appears to protect enamel 123 .In addition, milk contains calcium and calcium lactate, which may have a preventive effect against caries 124 , and can reduce the colonization of pathogenic microorganisms 125 .Furthermore, milk and cheese promote the dominance of casein phosphopeptides which are known to play a key role in biomineralization 126 .A systematic review and meta-analysis showed that dairy products containing probiotics had a significant impact in reducing S. mutans and raising salivary pH 122 .
Interestingly, the slow release of probiotics can also be achieved using appropriate embedding materials.For instance, L. paracasei 28.4-gellan formulations were recently found to release the probiotic for over 24 h 127 .L. paracasei in this state was able to inhibit S. mutans in both the floating and biofilm states, significantly reduce the generation of EPS, and downregulate the luxS, brpA, gbpB, and gtfB genes.

Controversy
Some researchers have taken a critical view of the idea that probiotics can prevent dental caries, primarily focusing on its safety and potential cariogenic effects.The majority of probiotics are not derived from the oral microbiota but from fecal samples, and some even come from animals 82 .Therefore, it is necessary to thoroughly evaluate their safety before clinical use.Probiotics may pose a risk to individuals with damaged barriers or low immunity, such as bacteremia 128 because high concentrations of administration are key to medication.Regarding the cariogenic issue, after conducting a meta-analysis of 50 experiments related to dental caries and periodontal diseases, Gruner and colleagues concluded that there is insufficient evidence to support the use of probiotics in treating dental caries 129 .Subjects with active dental caries showed higher levels of S. mutans, Actinomyces sp.strain B19SC, and Lactobacillus spp. as detected by PCR-based methods 130 .The major lactic acid bacteria identified from carious lesions, including both adults and children, include L. fermentum, L. casei/paracasei, L. salivarius, L. rhamnosus, L. plantarum, and L. gasseri 131 .Bifidobacterium dentium, considered a late marker for dental caries progression, was not found in the oral cavity of caries-free individuals but was detected in 30.8% of caries cases in a study of 56 participants 132 .
It is unreasonable to conclude a causal relationship between lactic acid bacteria and caries if there is a strong correlation between lactic acid bacteria and caries scores 133 .Lactic acid bacteria have a relatively low affinity for teeth, and their ability to form biofilms in vitro is much weaker than that of S. mutans 131 .The attachment and proliferation of secondary invaders including Bifidobacterium and lactic acid bacteria requires initiation of caries by major caries promoters including S. mutans to create an anaerobic acidic environment rich in carbohydrates 132,133 .The destruction of dentin is not enough by lactic acid alone but also requires proteolytic activity, because the main component of dentin 134 is more than the extracellular matrix dominated by type I collagen.However, lactic acid bacteria, including L. rhamnosus, L. casei/paracasei, L. salivarius, Lactobacillus vaginalis, Lactobacillus gasseri, Limosilactobacillus oris, and L. fermentum, have a greater propensity to bind to collagen rather than degrade it based on genomic analysis 133 .
In conclusion, the effectiveness of probiotics in preventing dental caries remains controversial.It is beneficial to prevent dental caries by exploring the roles of each microflora in the transition from oral health microbiota to cariogenic microbiota.In particular, from the perspective of oral microecology, the diet composition, host immune environment, and the physical and chemical characteristics of the oral cavity, especially the teeth, should be fully considered.Therefore, in recent years, researchers have become increasingly interested in exploring the benefits of prebiotics, synbiotics, and postbiotics in preventing dental caries, especially in terms of advantages over probiotics.

Prebiotics
In 1995, prebiotics were defined as "non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria already resident in the colon" 135 .However, with advancements in scientific research, ISAPP deemed the definition of prebiotics as "a substrate that is selectively utilized by host microorganisms conferring a health benefit" more appropriate in 2017 136 .Prebiotics present a safe and effective alternative to probiotic intervention since they are not live bacteria and are less susceptible to environmental factors affecting probiotic survival and efficacy.The following section will discuss prebiotics according to their different types, including their mechanisms of action, efficacies, and other related aspects.

Sugar
Interestingly, certain sugars exhibit prebiotic properties.D-tagatose, a noncariogenic sugar, is a potential prebiotic that offers lower calories and a lower glycemic index than sucrose 137 .Notably, the saliva of individuals with good oral health is rich in D-tagatose 138 .D-tagatose may inhibit the growth of S. mutans and S. gordonii by affecting glycolysis and its downstream metabolism, but it does not affect S. oralis 138 .Encouragingly, chewing gum containing D-tagatose has been shown to inhibit the growth of S. mutans 139 .
In addition to D-tagatose, other sugars such as xylose and arabinose are considered potential prebiotics, with the capacity to not only inhibit the growth of S. mutans but also promote the growth of Lactobacillus 140 .This dual action is particularly advantageous, as it may help restore the balance of the oral microbiome.Given the promising prebiotic properties of these sugars, further research is needed to assess their effectiveness in vivo and their potential side effects.

Sugar alcohol
Sugar alcohols, such as xylitol, sorbitol, maltitol, and erythritol, have been shown to exhibit prebiotic properties that can enhance oral health.Xylitol, a five-carbon polyol sweetener 141 , is considered an oral-specific prebiotic according to the new definition established in 2017 142,143 .It offers numerous benefits, including the enhancement of remineralization, decrease of the pH of dental plaque, reduction of the level of S. mutans in saliva, reduction of the insoluble dextran in the biofilm of S. mutans, and reduction of dental caries incidence [144][145][146] .However, xylitol loses its effect in the presence of fructose or sucrose 147 .Other sugar alcohols, such as sorbitol, maltitol, and erythritol, have also been shown to inhibit dental caries 148,149 .

Oligosaccharides
In addition to sugar alcohols, oligosaccharides are also being investigated as prebiotics.Human milk oligosaccharides (HMOs), the third most abundant ingredient in human milk, are often added to infant formula 150 .Galactooligosaccharides (GOS) and 2′-fucosyllactose, the most abundant HMOs, were found to reduce the EPS-mediated adhesion of S. mutans DSM 20523 to the glass surface, indicating their potential as prebiotics for oral health promotion 150 .In addition, GOS, glucomannan hydrolysates, and mannose have also been shown to inhibit pathogen adhesion to epithelial cells by binding to the pathogen's lectins/pili 151 .However, it should be noted that non-digestible and/or non-absorbable sugar alcohols and oligosaccharides, though beneficial for health, excessive intake may lead to significant diarrhea 152 .Therefore, further research is needed to determine the optimal dosage and duration of intake to minimize such adverse effects.

Arginine
Arginine is a widely studied oral prebiotic that has been shown to exhibit various beneficial effects on oral health 38 .These benefits include the promotion of alkaline substance production, the mitigation of tooth demineralization, and the suppression of biofilm formation.Specifically, arginine can inhibit the growth of Candida 153 and reduce enamel demineralization 154 .Furthermore, L-arginine has been found to enhance the alkali-producing ability of arginine-solubilizing bacteria, such as Streptococcus sanguinis and S. gordonii 155 , thereby making the biofilm environment unsuitable for cariogenic microflora by increasing the pH.Interestingly, L-arginine was found to significantly reduce the amount of insoluble EPS by 3-fold, targeting gtfB 156 .
Numerous studies have used toothpaste containing arginine to explore the mechanism underlying its prebiotic effects on oral health in depth.An in vivo study conducted on the oral ecosystem has revealed that the presence of arginine in toothpaste enhances the arginolytic capacity of human saliva while reducing its sucrose metabolic activity 157 .Additionally, it promotes a shift in the composition of salivary microbiota towards a healthier ecological state.Notably, tubes of toothpaste containing arginine and fluoride were more effective at preventing and reversing early caries lesions 158 and significantly increased re-mineralization compared with fluoride-only toothpaste 159 .Using toothpaste containing fluoride and arginine was also associated with an increase in gene expression associated with the arginine deiminase pathway, according to metagenomic and metagenomic data 160 .Moreover, the use of toothpaste containing fluoride and arginine was found to reduce caries bacteria and promote healthier microbial communities.
Interestingly, arginine may stimulate the production of effective substances by probiotics.Exogenous arginine has been found to increase the expression of the S. gordonii spxB gene, which encodes a pyruvate oxidase (SpxB), thereby promoting hydrogen peroxide production 156 .Additionally, using Mg 2+ as a cofactor of the catalytic activity of SpxB 161 has been shown to increase the production of H 2 O 2 and promote the abundance of SpxB in S. sanguinis and S. gordonii 161 .

Urea and nitrates
Urea and nitrates have been investigated as potential oral prebiotics.Urea is considered a prebiotic 162 due to its ability to be converted into ammonia or ammonium and bicarbonate ion by bacteria possessing urease, such as S. salivarius, Actinomyces naeslundii, and Haemophilus spp., thereby neutralizing acids in the oral cavity 163 .Nitrate-reducing bacteria in the oral cavity convert salivary nitrate to nitrite, which is subsequently reduced to nitric oxide 164 .All three compounds have been shown to restrict the growth of pathogenic bacteria 165,166 .Nitrate has demonstrated the ability to reduce caries incidence 164,165 and inhibit bacteria commonly associated with dental caries, such as S. mutans NCTC 10499, L. casei, and A. naeslundii, as well as periodontal disease-related bacteria including F. nucleatum, Eikenella corrodens, and Porphyromonas gingivalis 167 .However, it can lead to an increase in the levels of Neisseria and Rothia, genera associated with oral health and nitrate reduction 168 .Salivary nitrate supports nitrate respiration by anaerobic microorganisms, ultimately increasing oral pH through various mechanisms.These mechanisms include competition for carbon sources with acid-producing fermentation processes, generation of hydroxyl ions, and dissimilation of nitrate into ammonium to consume organic acids 168,169 .In addition to its impact on dental caries, dietary nitrate not only benefits oral health by significantly reducing gum inflammation 170 but also contributes to overall health by lowering systemic blood pressure 171,172 , promoting vascular health 172 , and even potentially improving vascular function in patients with hypercholesterolemia 173 .Considering the systemic health effects of nitrate, further investigation is warranted to explore other mechanisms through which nitrate can prevent dental caries.
Both probiotics and prebiotics have beneficial effects on health and combining them appropriately for co-administration represents another approach to drug administration.The following section will further elaborate on this combination.

Synbiotics
The combined use of probiotics and prebiotics has demonstrated superior therapeutic effects compared to their utilization 174 .The term "synbiotics" was first coined in 1995 by Gibson et al. to describe the combination of probiotics and prebiotics 135 .ISAPP updated the definition of synbiotics in 2020, stating that it is a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host 175 .It is not surprising that synbiotics have received less attention compared to probiotics, as it was proposed relatively later.
Several studies have indicated potential oral health benefits associated with synbiotic use.For instance, Nunpan et al. 176 demonstrated that the synbiotic composed of L. acidophilus in combination with GOS and fructooligosaccharides can significantly inhibit the growth of S. mutans.Tester and Al-Ghazzewi 177 found that synbiotics composed of Konjac glucomannan hydrolysates and L. acidophilus reduced the levels of S. mutans in vitro.In another study, Kojima et al. proposed a novel symbiotic 140 .They screened five strains of lactobacilli using sugar assimilation tests with 12 different saccharides, among which the three most promising prebiotics were found to be arabinose, xylose, and xylitol.The selected lactobacilli significantly inhibited the production of water-insoluble glucan by S. mutans 140 .
Considering the significant role of excessive acid in dental caries development, the selection of synbiotics comprising prebiotics capable of maintaining a high pH oral environment represents an innovative and intelligent approach.Synbiotics composed of 2% L-arginine and L. rhamnosus not only reduced the biomass of S. mutans biofilm but also decreased lactate content in spent media, resulting in no significant decline in pH within 24 h 178 .This suggests that synbiotics modulate the ecology of dental plaque.Remarkably, this study also observed that the addition of L-arginine promoted the positive utilization of amino acid biosynthetic pathways by L. rhamnosus, thus facilitating its proliferation.These findings indicate that the selection of synbiotics capable of pH regulation may offer stronger advantages in the oral cavity.
These findings provide promising evidence for the development of synbiotics as a novel approach to improving oral health.Further research is needed to establish the safety and efficacy of different synbiotic combinations.It is hoped that these advances will lead to the development of innovative yet effective strategies for promoting oral health, thereby improving overall health outcomes.

Postbiotics
In 2021, ISAPP defined "postbiotics" as "the preparation of inanimate microorganisms and/or their components that confer a health benefit on the host" 179 , excluding essentially purified metabolites such as butyric acid 179 .Before this, the term and definition of postbiotics were not officially standardized and unified.Postbiotics have also been referred to as "paraprobiotics", "heat-killed probiotics", "ghost probiotics", "non-viable probiotics" and "bacterial lysates" 180 .Over the past few years, the research direction of postbiotics has garnered increasing attention from researchers and has gradually become a hot research topic.Postbiotics are considered superior to probiotics due to their good acid-base and thermal stability, ease of storage and use, and high safety, as many probiotics are sensitive to oxygen and heat 179,181 .The feature of postbiotics enables them to be added to common products such as toothpaste, chewing gum, natto, potato chips, popcorn, and suckable candies 179,182 .Another advantage of postbiotics is that microorganisms cannot be isolated from commercial products, thus enabling product developers to maintain ownership of their components 179 .
The main methods of preparing postbiotics are heat inactivation of bacterial cells and preparation of CFS.In addition, other inactivated https://doi.org/10.1038/s41522-024-00488-7 Review article npj Biofilms and Microbiomes | (2024) 10:14 technologies, such as electric field, ultrasonication, high pressure, X-rays, high voltage electrical discharge, magnetic field heating, moderate magnetic field, and plasma technology, are also available 179,183 .It should be noted that the mode of inactivation may affect the activity of postbiotics to some extent.For example, in one study, the activity of CFS of S. oralis subsp.dentisani 7746 concentrated at 60 °C was lost, while CFS concentrated at 30 °C or 45 °C retained its activity 82 .The ability of heat-killed L. reuteri to adhere to and inhibit pathogens was significantly reduced compared to L. reuteri 110 , possibly due to the alteration of physical and chemical properties caused by heat treatment 184 .
Although postbiotics do not contain live microorganisms, this does not imply that inactivated bacteria have completely lost all beneficial properties.For example, heat-killed Bifidobacterium animalis BB12 still can reduce the cariogenicity of biofilm in vitro 185 , indicating that heat-killed bacteria did not completely lose all their beneficial properties.As another example, L. paracasei DSMZ16671 maintained its ability to co-aggregate with S. mutans after heat-killed treatment (autoclaved at 121 °C for 20 min) 116 .There was even a study that showed that L. rhamnosus CNCM-I-3698 and Companilactobacillus farciminis CNCM-I-3699 had a greater ability to exclude pathogens and adhesion after heat-killed treatment 186 .The inactivation process may lead to the disruption of bacterial cell structures, making bioactive molecules more exposed and accessible for utilization 179 .Postbiotics, which are mixtures of various components, may exert their health effects on the oral cavity through multiple mechanisms.These mechanisms can be independent or cooperative and may resemble the previously described mechanisms of probiotic health effects.In the interest of brevity, we will not reiterate these mechanisms here.
In the context of dental caries prevention and treatment, the types of postbiotics commonly utilized include CFS and heat-killed probiotics.However, it is worth mentioning that research in fields beyond the oral cavity has explored a broader range of postbiotic types, such as peptidoglycans, lipopolysaccharides, and pili 179 .Additional investigations into the mechanisms by which other types of postbiotics act concerning dental caries have the potential to provide innovative approaches for the prevention and treatment of this condition.Furthermore, further studies are warranted to assess the safety and effectiveness of postbiotics, thereby contributing to the improvement of overall health outcomes.

Conclusion and future perspectives
Dental caries prevention and treatment is a critical issue in oral healthcare.The traditional prevention and treatment methods have mainly focused on physical removal and chemical inhibition.While minimally invasive dental restoration can treat cavities, it fails to address the underlying causes of new caries formation.Thus, safer, more effective, and personalized preventive and treatment strategies are urgently needed.Extensive research has been conducted on the application of microbial preparations, such as probiotics, prebiotics, synbiotics, and postbiotics, in the prevention and treatment of dental caries, with promising outcomes.These microbial preparations can modulate the balance of oral microbiota by introducing beneficial microorganisms or inhibiting pathogenic ones.This review aims to help overcome theoretical obstacles to the successful clinical application of microbial preparations in preventing and treating dental caries.
In conclusion, investigating the potential applications of microbial preparations in the prevention and treatment of dental caries is an essential research avenue in the field of oral microbiology.The successful application of microbial preparations in the clinical setting can provide crucial support and greater assurance for oral health.Dental caries pathogenesis is closely associated with the balance of oral microbiota.Therefore, future research should focus on obtaining a deeper understanding of the characteristics and interrelationships among various beneficial and harmful microbial populations.With such comprehensive research, it is advantageous to develop more effective microbial preparations and optimal allocation methods.Developing personalized treatment plans is the current trend, and by devising microbial preparations that are effective for different dental caries risk and medication risk populations, personalized prevention and treatment of dental caries can be achieved.Furthermore, personalized treatment schemes are not limited to single microbial preparations and can be used alongside other treatment modalities to enhance the overall therapeutic outcomes and achieve the goal of curing dental caries.Improving efficacy and safety is a critical direction for future research.Based on a further exploration of the mechanisms of action of microbial preparations, refining and optimizing formulations according to research findings can enhance the therapeutic effects and safety of microbial preparations.

Fig. 2 |
Fig.2| The mechanism of probiotics to prevent dental caries.It is roughly divided into five parts.A Production of active metabolites: probiotics directly inhibit cariogenic pathogens by active metabolites (e.g., bacteriocin, enzyme, biosurfactants, organic acids, and hydrogen peroxide), which themselves have bacteriostatic activity.B Inhibition of cariogenic microbial biofilm: probiotics can inhibit or remove the biofilm of oral cariogenic microorganisms.C Competitive adhesion and colonization: probiotics not only occupy the colonized sites in the oral cavity but also inhibit the adhesion ability of cariogenic microorganisms.D Coaggregation with pathogens: probiotics inhibit cariogenic microorganisms colonization in the oral cavity through co-aggregation.E Regulation of the immune system: probiotics activate or modulate the host immune system, thereby enhancing the immune response to cariogenic microorganisms (enhances salivary levels of human neutrophil peptides 1-3).

14 Table 1 | 187 Milk 188 Yogurt 32
https://doi.org/10.1038/s41522-024-00488-7Review article npj Biofilms and Microbiomes | (2024) 10:Application vehicle and therapeutic effects of probiotics of S. mutans in saliva and delayed the development of new dental caries nonsyndromic cleft lip and palate patients with a mean age of 19 reduced the count of S. mutans, while increasing the count of Lactobacillus and the colonization for 2 weeks 66 students aged 18-30 with initial stages of dental caries reduced the count of S. mutans and Lactobacillus in the probiotic group 191 Cheese L. acidophilus NCFM or L. rhamnosus Lr-daily for 2 weeks with breakfast and dinner meals 60 adults with a mean age of 28 could not reduce the count of S. mutans and Lactobacillus in the probiotic group 194 Ice cream B. lactis Bb-12 and L. acidophilus La-5 1-2 months no impact on the oral colonization of B. animalis lactis BB-12 and mutans streptococci in the early and B. lactis BB12 (Mother dairy b-activ Plus®) unclear once daily for 7 days before breakfast 60 caries-free adults aged 20-25 Significantly improved salivary pH and reduced the count of salivary S. mutans five times a week for 7 months 530 healthy children aged 3-6 reduced the count of S. mutans and the risk of dental aged 21-35 reduced the levels of S. mutans 202 CFU colony forming units, B. animalis Bifidobacterium animalis, S. uberis Streptococcus uberis, S. rattus Streptococcus rattus.https://doi.org/10.1038/s41522-024-00488-7Review article npj Biofilms and Microbiomes | (2024) 10:14